Compositions and methods for disease diagnosis using single cell analysis

ABSTRACT

Certain embodiments of the invention are directed to evaluating and identifying cells by recording and interpreting a time-dependent signal produced by unique cell respiration and permeability attributes of isolated viable cells.

PRIORITY CLAIM

This application is a continuation of U.S. application Ser. No.15/833,629 filed Dec. 6, 2017, which is a continuation of U.S.application Ser. No. 15/466,377 filed Mar. 22, 2017 which claimspriority to U.S. Application No. 62/407,311, filed Oct. 12, 2016, eachof which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This disclosure relates generally to cellular biology and moreparticularly to compositions and methods for rapid and sensitiveidentification of disease-causing cells and the rapid and sensitivecharacterization of their response to the drugs used to treat them.

BACKGROUND

In various instances, Disease-Causing Cells (DCCs) are found inquantities below the limit-of-detection of conventional analyticaltechniques. Thus, methods for identifying DCCs and characterizing theirresponse to treatment typically require either multiplication of thetarget cells and/or target-dependent amplification of the target cells'molecular contents and/or products, depending on the application.

Due to high sensitivity compared to other techniques, nucleic acidamplification (e.g., PCR-based) tests (NATs) have become the preferredmethod for fast pathogen identification. Culture based methods are thecurrently preferred method for antimicrobial susceptibility testingbecause the methods assess the microbial phenotype, providing highclinical validity.

NATs require complicated sample workflow steps which usually includecell lysis followed by nucleic acid concentration step that also removesPCR inhibitors from the samples. Cell lysis creates an asymmetry in therequirements for nucleic acid extraction efficiency. Mycobacteria andfungi, for example, possess a very thick cell wall compared togram-negative bacteria and thus are far more difficult to lyse, usuallyrequiring a mechanical lysis step to efficiently disrupt their cellwall. Consequently, NATs that utilize simple chemical lysis methodsoften lack sensitivity for these tougher pathogens. Furthermore, celllysis reagents are extremely inhibitory to PCR reaction because they aredesigned to efficiently denature proteins and PCR utilizes proteins toperform the amplification reaction. Cell lysis, therefore, introducesthe need for highly efficient wash steps to remove lysis reagents fromthe extracted nucleic acid. In addition, NATs require expensive assaydevelopment methods because they rely on pathogen-specific reportermolecules (primers and probes) that must be designed specifically foreach target. Each NAT must therefore include an expensive molecular R&Dprocess which involves primer/probe design and screening for eachtarget.

Furthermore, if a NAT assay includes more than one target (multiplexassay), requiring more than one reporter species (primer pair), thedifferent target and/or reporter species can interact nonspecificallywith one another, causing either false positives when the reporteramplifies nonspecifically against the other reagents or false-negativeswhen the target amplification reaction is inhibited by a non-specificinteraction with another species. Consequently, this limits how manytargets can be identified within a single NAT. This becomes particularlyrelevant with the issue of drug resistance because, in the case ofgram-negative bacteria and mycobacteria, there are numerous mutations(each mutation being a target) indicative of resistance. NATs can onlyinterrogate a small fraction of those mutations within a single test. Inaddition, the genes that reside in an organism's genotype may not alwayscontribute to the phenotype. Therefore, genotypic information oftenportrays an inaccurate or incomplete picture of a pathogen's phenotypicresponse. Methicillin-resistant Staphylococcus aureus (MRSA), forexample, often do not express the mecA gene that confers resistance.Therefore, when it comes to the clinically important determination ofwhether the pathogen causing an infection is susceptible to a particulardrug NATs have low clinical validity.

NATs that include two or more targets (multiplexed) cannotsimultaneously quantify those targets accurately and precisely. This isbecause the same nonspecific interactions between reporter speciesdescribed above also cause variances in the PCR signal output andquantitative PCR relies on reproducible reaction results in order tocorrelate the generated amplification curves to the initial targetconcentration. This is a significant limitation because it prevents theuse of multiplex NATs for the diagnosis of infections from non-sterilefluids since humans are often colonized by the same pathogens that cancause an infection. In non-sterile fluids, therefore, the number ofpathogens present in the clinical sample (the pathogen load) is whatdetermines whether a bacterial species is causing an infection or“peacefully” colonizing the fluid. For example, in order to definitivelydiagnose the source of a pneumonia infection from a bronchioscopicspecimen (e.g., bronchooalveolar lavage (BAL)) the pathogen load for anybacteria present in the sample must exceed 10³ CFU/mL to be consideredthe source of an infection. Similarly, for urine specimens the thresholdis 10⁵ CFU/mL.

In addition, quantitative culture methods encounter problems identifyingpolymicrobial infections when one of the infecting pathogens is afastidious organism (an organism that has a complex nutritionalrequirement and typically only grows under specific conditions) and theother is not. The non-fastidious organism will often drown out thefastidious organism on a culture plate and conceal its presence in thespecimen. This is particularly problematic because many of thedrug-resistant microbes are fastidious non-fermenting rods(gram-negatives).

Thus, there remains a need for additional methods and apparatus foridentifying and characterizing disease causing cells (DCCs) in a fastand/or efficient manner.

SUMMARY

Certain aspects of the methods described herein can be practiced usingan integrated workflow and analysis that can be automated and performedon disposable cartridge providing integrated testing without having toutilized one or more kits to perform sample preparation prior toanalysis. The methods described can be implemented as a single stepautomated process. The compositions and methods described herein addressthe various problems associated with the current methods for identifyingand characterizing DCCs. DCCs are defined herein as either host cells,such as malignant or disease-associated cells of the host from which thesample is taken (e.g., cancer cells), or acquired cells (e.g., fungal orbacterial), which include any microflora associated with, involved in,implicated in, or indicative of a disease or pathology. Such diseasesinclude, but are not limited to cancer and infections.

Embodiments of the invention are directed to methods that identifypathogens by recording and interpreting a time-dependent signal producedby unique cellular metabolism, respiration, and/or permeabilityattributes of isolated cells. In certain aspects of the currentinvention does not require lysis or washing. As used herein, metabolismrefers to the set of life-sustaining chemical transformations orprocesses within a cell. The three main purposes of metabolism are theconversion of food/fuel to energy to run cellular processes; theconversion of food/fuel to building blocks for proteins, lipids, nucleicacids, and some carbohydrates; and the elimination of nitrogenouswastes. Respiration as referred to herein is cellular respiration, a setof metabolic reactions and processes that take place in a cell ororganisms to convert biochemical energy from nutrients into adenosinetriphosphate (ATP), and then release waste products. Furthermore, sinceintact cells are used, only whole cells need to be manipulated ratherthan nucleic acid molecules, which are much more difficult manipulatedue to their small size and propensity for charge-based interactionswith different materials. Furthermore, the cells can be incubated at asingle temperature, typically a relatively low temperature in the rangeof 25 to 45° C., obviating the need for thermal cycling equipmentrequired for most NATs, which reduces cost and workflow complexity ofthe currently described invention. Advantageously, by avoiding hightemperature steps, the invention avoids significant issues that canarise with fluid evaporation and/or bubbles that can disrupt theintegrity of the reaction and/or the fluorescent readout.

Furthermore, certain methods described herein utilize a single,universal reporter molecule for all target cells. As used herein areporter is a molecule, peptide, protein, or other compound that variesin fluorescence emission, absorption, and/or reflectance in relation toa variation in environment or condition, such as reduction-oxidation(redox) state. Thus, unlike NATs, where each reporter molecule isdesigned to respond only to one particular target, the universalreporter in the described methods responds to all the targets (e.g.,DCCs) in a target specific manner, that is the response can be differentfor each target using the same reporter. In certain instances it is the“shape” or waveform of the reporter signal over time that changes or isunique to a target rather than the reporter molecules used to generatethe presence or absence of a signal. Thus, it is the “system” that actslike a probe rather than the individual reporter molecules. With auniversal reporter, assay development becomes, in certain instances,effectively a software R&D exercise because only data storage andanalysis are changed between targets. This offers a significantadvantage because software changes are much faster and cheaper toimplement, augment, and test compared to molecular or chemistry changes.In some cases, it may be necessary to change the cell suspensionformulation to include certain drugs or nutrients, formulation changesthat are much faster and simpler to implement and optimize thanmolecular design changes.

The methods described herein produce metabolic, respiratory, ordrug-susceptibility profiles that can be used to identify or phenotype atarget cell in a sample. Specifically, just as this method uses amammalian cell's or microbial cell's unique metabolism, respiration,and/or permeability characteristics to distinguish between differentcellular and microbial species, those same characteristics can be usedto determine whether the cell or microbe is susceptible to a particularcompound, cytotoxic compound, or antimicrobial, since a compound or aneffective drug will alter the cellular metabolism, respiration, and/orpermeability characteristics. In certain aspects methods of theinvention can interrogate the interactions of a compound(s) orconditions on a cell, be it (i) a normal cell for determining toxicityof a compound or condition, or (ii) a pathogenic or disease-related cellfor determining therapeutic efficacy of a compound or condition. Thismethod can account for all resistance mechanisms that may conferresistance to a particular cell and, therefore, offers higher clinicalvalidity than that of NATs, which can only account for a fraction of theresistance mechanism. This is why, despite being much faster, NATs havenot been able to replace culture-based methods for drug-susceptibilitytesting. NATs typically produce a result in about an hour whereasexisting culture-based drug-susceptibility methods typically requireover 48 hrs.

The currently described methods produce phenotypic susceptibilityresults at significantly faster turn-around-times (<4 to 6 hrs) thanculture-based phenotypic methods. The increase in speed compared toculture is accomplished, in part, by the rapid signal concentration madepossible at sub-nanoliter volumes which are orders of magnitude smallerthan the milli- and microliter volumes typically used by other methods.Put simply: confinement of each cell or microbe into sub-nanoliterdroplets enables reporters (e.g., fluorescent molecules) to be rapidlydetected or rapidly concentrate to detectable levels. And since thismethod can detect single cells, the method is as sensitive as NATs whichare more sensitive the culture methods. Thus, the described methodsoffers the analytical validity of NATs combined with the clinicalvalidity of culture based methods.

In the method described herein, individual cells or microbes areisolated or partitioned into separate droplets, enabling quantificationto become the simple matter of counting those droplets that produced asignal indicating the presence of a target cell or microbe. The shape orchange of the reporter signal over time (e.g., a waveform), which is oneof the characteristics relied upon for pathogen identification andcharacterization, is orthogonal to the method for quantifying the numberof pathogens—one does not affect the other. Thus, accurate and precisemultiplexed identification and quantification is accomplishedsimultaneously.

Finally, the reporter or cell viability reagents used in the inventionare inexpensive compared to the materials used in NATs. And because oftheir simple structure, they can be easily lyophilized and solubilized.A variety of reporters may be used with the systems and methodsdisclosed herein. For example, the at least one small molecule metabolicreporter can be a fluorophore, a protein labeled fluorophore, a proteincomprising a photooxidizable cofactor, a protein comprising anotherintercalated fluorophore, a mitochondrial vital stain or dye, a dyeexhibiting at least one of a redox potential, a membrane localizing dye,a dye with energy transfer properties, and/or a pH indicating dye. In afurther aspect the reporter can be or include a resazurin dye, atetrazolium dye, coumarin dye, an anthraquinone dye, a cyanine dye, anazo dye, a xanthene dye, an arylmethine dye, a pyrene derivative dye, aruthenium bipyridyl complex dye or a derivative thereof. As used herein,“derivative” is understood to mean a chemically modified form of a dyethat maintains some of the detection characteristics, e.g., fluorescentsensitivity to redox state, of the compound from which it is derived.Certain embodiments utilize a resazurin-based dye which is veryinexpensive compared to PCR reagents, such as expensive enzymaticcomponents.

Some compounds or antimicrobials are only effective against certaincells or microbes (indeed, this is why, in the absence of definitivedrug-susceptibility information, it is important to identify thecausative pathogen to guide therapy), for example, a microbe's responseto a particular antimicrobial aids in identifying the microbe. Thissensitivity is one reason bacterial culture methods utilize antibioticsin the growth media to identifying the growing bacteria. Certainembodiments of the invention provide a method for rapid and sensitiveidentification of disease-causing cells and the rapid and sensitivecharacterization of their response to the drugs used to treat them. Incertain aspects, target cells are re-suspended with a reporter (e.g., aresazurin-based dye), poising agents or test compounds (optional), andcell nutrients (e.g., growth media), the suspension is compartmentalizedinto droplets and organized into a two-dimensional array where they areincubated and their fluorescence is monitored over time using an imagingsystem. Each viable DCC contained within a droplet will influence theenvironment or conditions in the drop which in turn affects thereporter. For instance, a viable DCC contained within a droplet canirreversibly reduce resazurin into highly fluorescent resorufin, causingthe droplets to emit fluoresce when excited. Subsequently, if theenvironmental oxidation-reduction (redox) potential dips below a certainthreshold, the resorufin is reversibly reduced to hydroresorufin, whichis a non-fluorescent molecule. If the redox potential rises above thesame certain threshold, the hydroresorufin is oxidized back to resorufinand fluoresces again. Thus, the amount of fluorescence emitted from adroplet can undulate over time depending on the changing environment orconditions (e.g., redox potential) of the droplet. Because cells havedifferent metabolic or redox characteristics, cells will producecharacteristic fluorescent undulations (signatures) that can be used toidentify which cell type is in the droplet. A characteristic signaturecan also be generated by stressing the cell with an environmentalstressor or condition, such as an antimicrobial drug or potentialtherapeutic. By combining the information contained within one or moreof these signatures, single cells contained within each droplet can beidentified and characterized.

In one embodiment, the cells in a test sample are divided into twopopulations, one population (the test population) includes anenvironmental stressor or test condition, and in the other population(the control population) the environmental stressor or test condition isexcluded. The two populations are observed over time. The cells in thetest sample may be identified by population characteristics and/or fromthe signatures in a control population. Identification may be furtheraided by the signatures generated in the test population, but theprimary purpose of the test population is to characterize the responseof the cells to the environmental stressor or test condition bycomparing the test population signatures to the control signatures.

Particular embodiments are directed to methods for identifying andcharacterizing a disease causing cell (DCC) in a sample or diagnosing adisease associated with a disease causing cell. The methods can comprise(a) contacting a sample suspected of containing one or more targetdisease causing cells (DCCs) with at least one reporter (e.g., aviability or reporter dye) forming a sample mixture; (b) partitioningthe sample mixture into partitions comprising at most one target DCC ornatural DCC aggregate per partition; (c) incubating the partitions for aperiod of time at a specified temperature or series of temperatures; (d)monitoring optical characteristics of the partitions during theincubation time; (e) constructing a waveform (signal over time) for eachpartition based on the optical characteristics over time; and (f)evaluating the sample using information provided by partition waveforms.As used herein, a natural DCC aggregate is an association of two or morecells having the same phenotype (i.e., a homogenous aggregate), theassociation of which is not readily dissociated by the processingconditions. In certain instances the DCC aggregate is an aggregate ofmicrobes having the same phenotype.

In certain aspects the partitions are droplets in an immiscible fluid.Upon mixing of the target-containing solution and the immiscible fluid,they form phases—an aqueous drop or partition, which holds the targetmaterial in solution, and a non-aqueous phase made up of the immisciblefluid. The immiscible fluid can be a fluorocarbon comprising afluorosurfactant or hydrocarbon oils such as mineral oil, or siliconeoils. In particular aspects the droplets can be between 0.1 pL and 10nL. In a further aspect the droplets are at least, at most, or about0.1. 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pL to 200,300, 400, 500, 600, 700, 800, 900, or 1000 pL, including all values andranges there between. In certain aspects the droplets are about 40 to300 pL. The methods can further comprise arranging the droplets in atwo-dimensional array. In certain aspects the two-dimensional array is astatic two-dimensional array. Partitioning of the sample can be done byLaplace pressure gradients or by using shear stress methods, as well asother methods for drop or partition formation.

Monitoring of the optical characteristics of the partitions can beperformed using a detector, such as a camera or the like. In certainaspects the optical characteristics include fluorescence of thepartition(s). In certain aspects the monitoring of the opticalcharacteristics of the partitions further comprises illuminating orexposing the partition with electromagnetic radiation, such as light. Incertain aspects the electromagnetic radiation comprises an excitationwavelength that is compatible with the reporter, i.e., illuminating orirradiating a partition with an appropriate source. In certain aspectsthe source provides light including an excitation wavelength of 500,525, 550, 575, 600, 625, 650, 675, to 700 nm, including all values andranges there between. The source will be selected so that theelectromagnetic radiation excites one or more reporter in the samples,e.g., dyes or other compounds. In particular aspect the light source canbe a light emitting diode (LED).

Reporters can include a “viability dye” or “reporter dye”, the viabilityor reporter dye is a moiety that detects changes in the environmentsurrounding an isolated cell due to a cell's viability, respiration, ormetabolic activity; or is a detectable protein that is expressed underspecific conditions (e.g., green fluorescent protein or luciferase). Incertain aspects a cell can be transfected or engineered to express areporter protein. The reporter can be detected using any method known inthe art appropriate to the reporter employed, for example light emissionor absorbance of a fluorophore or a colorimetric dye. In certaininstances, the signal from the reporter is detected by opticalmicroscopy, camera, or other detector/sensor as appropriate. In certainaspects the reporter is a fluorescent dye. In certain aspects thereporter is resazurin, a resazurin-based dye, or a dye that is aderivative of or structurally related to resazurin(7-Hydroxy-3H-phenoxazin-3-one 10-oxide). Resazurin is a non-toxic, cellpermeable compound that, in its oxidized state, is blue in color andvirtually non-fluorescent. When in contact with living cells, resazurinis reduced to resorufin, a compound that is red in color and highlyfluorescent, and can be detected fluorimetrically or colorimetrically.Metabolic activity of viable cells continuously convert resazurin toresorufin, increasing the overall fluorescence and color of the mediasurrounding cells. A resazurin-based dye is a dye that contains aresazurin structure in addition to other modifying groups. In otheraspects a viability dye is tetrazolium, a tetrazolium-based dye, or adye that is a derivative of or structurally related to tetrazolium. Atetrazolium-based dye is a dye that contains a tetrazolium moiety andmay contain other modifying groups that do not disrupt the five memberedtetrazolium ring.

In certain aspects the incubating of the partitions is at a constanttemperature (isothermal). In other aspects the temperature can becontrolled and can be stepped or ramped up using a particular intervalor rate, such as stepping up from 25 to 37° C. or increasing at a rateof 2 to 10° C. per minute. In still other aspects temperature can bedecreased at a particular interval or rate, such as decreasing at aninterval of 5 to 10° C. or a rate of 2 to 10° C. per minute. In variousaspects the temperature(s) are in the range of 20 to 45° C., 30 to 40°C., or 35 to 38° C., including all values and ranges there between. Incertain aspects partitions are incubated at 37° C.

In particular aspects the partitions comprise a single cell, microbe, orcellular or microbial aggregation. In a further aspect the partition maycontain 2, 3, 4, or more cell or microbe types. The methods can furthercomprise classifying a microbe by species, genus, family, order, class,phylum, kingdom, or a combination thereof. The classification can bebased on the characteristics of one or more waveforms under one or moreconditions. In certain aspects the microbe is a bacteria. Certainaspects of the invention can include classifying the bacteria bygram-stain group or other classification criteria recognized formicrobes, including bacteria. In certain instances a partition maycontain more than one target type (species, genus, etc.) but that anenvironmental stressor or condition may kill all but one target type,which can be identified using its signature or waveform.

The methods can further comprise dividing the sample into a controlsample and at least one test sample prior to partitioning. Each testsample can be treated or processed in a manner that differs from thecontrol. In certain aspects at least one test sample is contacted with astressor, cytotoxic, anticancer, antimicrobial compound or condition. Incertain aspects individual test samples can be exposed to differentconcentrations compounds or variations in conditions. In other aspects,a test sample can be exposed to a variety of temperatures, environments,or chemicals that may or may not alter the phenotype of the cellscontained in the test sample. In certain aspects a DCC is a pathologicor pathogenic cell, such as a cancer cell.

The methods include evaluating the sample (control and test samples)using the partition waveform (i.e., signal detected over time). Incertain aspects evaluating includes comparing the partition waveform toa library of stored or predetermined waveforms (e.g., waveformreference).

Certain embodiments are directed to methods for detecting andcharacterizing DCCs, such as microbes or cancer cells, in a samplecomprising (a) contacting a sample comprising microbes with a reporter,e.g., a viability dye, forming a sample mixture; (b) dividing the samplemixture into at least two portions or samples that include a controlsample and at least one test sample; (d) introducing a testcompound/substance or an antimicrobial drug to the at least one testsample; (e) partitioning each of the control sample and at least onetest sample into partitions forming control sample partitions and testsample partitions, wherein the partitions comprise at most one targetmicrobe or a natural aggregation of microbes; (f) incubating thepartitions over time at a specific temperature or temperatures; (g)monitoring optical characteristics of the partitions during theincubation time, wherein the optical characteristics include the amountof optical signal produced by interaction of the reporter with themicrobe in the partition; (h) constructing an optical signal waveformfor each partition resulting in a partition waveform; (i) classifyingthe microbe within each partition using the partition waveform shape;and (j) comparing partition waveforms between the control samplepartition waveforms and the test sample partition waveforms andassessing test compound/substance or antimicrobial drug susceptibilitybased partition waveform comparison. In certain aspects a test compoundcan include a small molecule, peptide, a nanoparticle, or a protein. Instill other instances a test substance can include bacteriophage andother engineered therapeutics. In other aspects the testcompound/substance can be a therapeutic identified as a therapy orengineered as a therapy for other disease conditions, such as cancer(e.g., chemotherapeutic or anti-cancer compound or substance).

Another primary application of this invention is towards the diagnosisand treatment of diseases such as cancer. Cancer diagnosis is verysimilar to infectious disease diagnosis in that disease causing cellscan mutate rapidly and can develop resistance to the drugs used to treatthem. Furthermore, cancer cells exhibit different morphological,metabolic, and respiratory characteristics than healthy cells.Specifically, cancer cells are known to exhibit differentoxidation-reduction characteristics from healthy cells. Furthermore,cancer cells are often much larger than healthy cells which alsoinfluences the amount of fluorescence generated during respiration.Because, certain aspects of this method are sensitive tooxidation-reduction (redox) changes that occur during cellularrespiration, those differences, as well as others, may be used todistinguish cancer cells from healthy cells. In addition, cancer cellsoften bypass apoptosis pathways and are therefore able to remain viablefor longer periods of time when they no longer reside in specifictissues. In this case, the cellular respiration would last longer forcancer cells compared to healthy cells, which could be observed assignal accumulates for longer periods of time for cancer cells comparedto healthy cells. In certain aspects the waveforms for normal cells willdifferentiate them from pathogenic or cancer cells.

As used herein, the term “partition” refers to a volume of fluid (e.g.,liquid or gas) that is a separated portion of a bulk volume. A bulkvolume may be partitioned into any suitable number (e.g., 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, etc.) of smaller volumes or partitions. Partitions may beseparated by a physical barrier or by physical forces (e.g., surfacetension, hydrophobic repulsion, etc.). Partitions generated from thelarger volume may be substantially uniform in size (monodisperse) or mayhave non-uniform sizes (polydisperse). Partitions may be produced by anysuitable manner, including emulsion, microfluidics, and microspraymethods. One example of partitions are droplets.

As used herein, the term “droplet” refers to a small volume of liquidwhich is immiscible with its surroundings (e.g., gases, liquids,surfaces, etc.). A droplet may reside upon a surface, be encapsulated bya fluid with which it is immiscible, such as the continuous phase of anemulsion, a gas, or a combination thereof. A droplet is typicallyspherical or substantially spherical in shape, but may be non-spherical.The shape of an otherwise spherical or substantially spherical dropletmay be altered by deposition onto a surface. A droplet may be a “simpledroplet” or a “compound droplet,” wherein one droplet encapsulates oneor more additional smaller droplets. The volume of a droplet and/or theaverage volume of a set of droplets provided herein is typically lessthan about one microliter, for example droplet volume can be about 1 μL,0.1 μL, 10 pL, 1 pL, 100 nL, 10 nL, 1 nL, 100 fL, 10 fL, 1 fL, includingall values and ranges there between. The diameter of a droplet and/orthe average diameter of a set of droplets provided herein is typicallyless than about one millimeter, for example 1 mm, 100 μm, 10 μm, to 1μm, including all values and ranges there between. Droplets may beformed by any suitable technique, including emulsification,microfluidics, etc., and may be monodisperse, substantially monodisperse(differing by less than 5%), or polydisperse.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. Each embodiment described herein is understood to be embodimentsof the invention that are applicable to all aspects of the invention. Itis contemplated that any embodiment discussed herein can be implementedwith respect to any method or composition of the invention, and viceversa. Furthermore, compositions and kits of the invention can be usedto achieve methods of the invention.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

In the following discussion and in the claims, the terms “including” and“comprising” are used in an open-ended fashion, and thus should beinterpreted to mean “including, but not limited to . . . .” Also, theterm “link” or “points to,” and forms thereof, are intended to meaneither an indirect or direct connection. Thus, if a first componentlinks to a second component, that connection may be through a directconnection or through an indirect connection via other components andconnections.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present disclosure, and theadvantages thereof, reference is now made to the following descriptionstaken in conjunction with the accompanying drawings, in which:

FIG. 1 is a schematic of a preferred embodiment of the invention appliedto generally to disease causing cells.

FIG. 2 is a schematic of a preferred embodiment of the invention whichas a method pathogen identification and antimicrobial susceptibilitytesting by monitoring the fluorescence over time of individual cellsand/or monoclonal cell clusters isolated in subnanoliter droplets with aredox-sensitive viability dye.

FIG. 3 is an illustration of a two-dimensional droplet array.

FIG. 4 is an illustration of a droplet monolayer being imaged by acamera. The droplet monolayer provides for good thermal conductivity andtemperature control.

FIG. 5 is an image a droplet array containing E. coli cells.

FIG. 6 is a schematic providing one explanation of as to fluorescencevariation according to the redox environment established by an isolatedcell.

FIG. 7 illustrates a waveform derived from an E. coli cell in a 268 pLdroplet.

FIG. 8 illustrates multiple waveforms from an array comprising amultiple cell types (E. coli and S. epidermis) partitioned intomicrodroplets. The waveforms are clearly distinguishable.

FIG. 9 provides a comparison of fluorescence relative to volume. Thefluorescence waveform generated by a single bacterium incubated inpico-liter droplets rises much faster than in larger volumes, asexpected, but also unexpectedly collapses due to a different redoxpotential in the picodroplet.

FIG. 10 illustrates one method of using shear stress for dropletgeneration.

FIG. 11 illustrates two methods of using Laplace pressure gradients fordroplet generation.

FIG. 12 illustrates the distinction between waveform monitoring ascompared to a discreet value at particular time. At pico-liter volumes,where the redox potential causes fluorescence to vary depending on thebacterial species, traditional endpoint analysis does not providedistinguishable viability information.

FIG. 13 illustrates that waveform information provides an increasedsensitivity when detecting drug-susceptibility.

FIG. 14 illustrates stored waveforms representing four bacterial speciesand their correlation to observed waveforms from the same species(median correlation coefficient) and, thus, can be used to identify thebacteria that generated the observed waveforms. Stored waveforms alsocorrelate well according to gram stain gram (B. subtilis and S.epidermidis are gram positives, E. coli and S. maltophilia are gramnegatives).

DETAILED DESCRIPTION

The following discussion is directed to various embodiments of theinvention. The term “invention” is not intended to refer to anyparticular embodiment or otherwise limit the scope of the disclosure.Although one or more of these embodiments may be preferred, theembodiments disclosed should not be interpreted, or otherwise used, aslimiting the scope of the disclosure, including the claims. In addition,one skilled in the art will understand that the following descriptionhas broad application, and the discussion of any embodiment is meantonly to be exemplary of that embodiment, and not intended to intimatethat the scope of the disclosure, including the claims, is limited tothat embodiment.

The invention generally relates to methods for disease diagnosis usingsingle-cell analysis. The following sections discuss generalconsiderations for test samples, compartmentalization/partitioning, cellviability and viability or reporter dyes, disease-causing cellaggregates/aggregation of microbes, signal detection, and multiplexing.

A general scheme is shown in FIG. 1. A test sample comprising at leastone target cell is combined with a viability or reporter dye andpartitioned into droplets such that a statistically significant numberof droplets contain no more than one target cell or aggregation of cells(some microbial species tend to aggregate into cell clusters or chains).In a preferred embodiment, a viability or reporter dye will be reducedfrom a non-fluorescent molecule to a fluorescent molecule in thepresence of a viable cell and then further reduced to a non-fluorescentmolecule if the redox potential in the droplet drops below a certainamount, typically −100 mV. The fluorescent signature generated in eachdroplet is monitored over time and used to identify and characterize thecell contained within. Further details on the processes of the inventionare provided below.

Test Sample.

Target cells in the test sample include bacteria, fungi, plant cells,animal cells, or cells from any other cellular organism. The cells maybe cultured cells or cells obtained directly from naturally occurringsources. The cells may be obtained directly from an organism or from abiological sample obtained from an organism, e.g., from sputum, saliva,urine, blood, cerebrospinal fluid, seminal fluid, stool, and tissue. Anytissue or body fluid specimen. In one embodiment the test sampleincludes cells that are isolated from a biological sample comprising avariety of other components, such as non-target cells (backgroundcells), viruses, proteins, and cell-free nucleic acids. The cells may beinfected with a virus or another intracellular pathogen. The isolatedcells may then be re-suspended in different media than those from whichthey were obtained. In one embodiment the test sample comprises cellssuspended in a nutrient medium that enables them to replicate and/orremain viable. The nutrient media may be defined media with knownquantities or all ingredients or an undefined media where the nutrientsare complex ingredients such yeast extract or casein hydrolysate, whichcontain a mixture of many chemical species of unknown proportions,including a carbon source such as glucose, water, various salts, aminoacids and nitrogen. In one embodiment, the target cells in the testsample comprise pathogens and the nutrient media comprises a commonlyused nutrient broth (liquid media) for culturing pathogens such aslysogeny broth, nutrient broth or tryptic soy broth. In any embodimentthe media may be supplemented with a blood serum or synthetic serum tofacilitate the growth of fastidious organisms.

Compartmentalization/Partitioning.

The methods of the invention involve combining a test sample comprisingat least one target cell with a viability or reporter dye and thenpartitioning the test sample into droplets such that no droplet containsmore than one target cell or cell aggregates. The number of droplets canvary from hundreds to millions depending on the application and dropletvolumes can also vary between 1 pL to 100 nL depending on theapplication, but preferably between 25-500 pL. The methods describedherein are compatible with any droplet generation method. Exemplarymethods for droplet generation are shown in FIG. 10 and FIG. 11. Whilethe methods for droplet formation differ, all the methods disperse anaqueous phase, the test sample in this case, into an immiscible phase,also referred to as the continuous phase, so that each droplet issurrounded by an immiscible carrier fluid. In one embodiment theimmiscible phase is an oil wherein the oil comprises a surfactant. In arelated embodiment, the immiscible phase is a fluorocarbon oilcomprising a fluoro-surfactant. An important advantage to using afluorocarbon oil is that it is able to dissolve gases relatively welland it is biologically inert. Thus, the fluorocarbon oil used in themethods described herein comprises solubilized gases necessary for cellviability.

One non-limiting example of partition formation is by using Laplacepressure gradients (see, for example, Dangla et al., 2013, PNAS110(3):853-58). Laplace pressure is the differential pressure betweenthe inside and outside of a curved surface, such as the difference inpressure between the inside and outside of a droplet. An aqueous phasecontaining cells or microbes can be introduced into a device having areservoir of a continuous phase (i.e., immiscible fluid) forming anaqueous “tongue” in an appropriate device. The device can incorporateheight variation(s) into a microchannel that subject the immiscibleinterfaces to a difference in curvature between the portion of theaqueous phase that has not encountered the height variation and theportion of the aqueous phase downstream of the height variation. As theaqueous phase flows through the height variation, a critical curvatureis reached for the portion of the aqueous phase downstream of the heightvariation beyond which the two portions cannot remain in staticequilibrium, breaking of the aqueous phase into a droplet, as thedownstream portion detaches from the tongue formed by introduction ofthe aqueous phase into a continuous phase, the size of the drops beingdetermined by the device geometry. The height variation can beaccomplished with a single step change in the height of a microchannel(step emulsification), multiple steps (multi-step emulsification), and aramp or similarly gradual gradients of confinement.

Reporters.

A variety of reporters may be used with the systems and methodsdisclosed herein. For example, the at least one small molecule metabolicreporter can be a fluorophore, a protein labeled fluorophore, a proteincomprising a photooxidizable cofactor, a protein comprising anotherintercalated fluorophore, a mitochondrial vital stain or dye, a dyeexhibiting at least one of a redox potential, a membrane localizing dye,a dye with energy transfer properties, a pH indicating dye. In a furtheraspect the reporter can be or include a resazurin dye, a tetrazoliumdye, coumarin dye, an anthraquinone dye, a cyanine dye, an azo dye, axanthene dye, an arylmethine dye, a pyrene derivative dye, a rutheniumbipyridyl complex dye or derivatives thereof. Certain embodimentsutilize a resazurin-based dye. Cell viability dyes, which are alsoincluded in the term reporter used herein, are used as analysis reagentsto identify and characterize individual cells or pathogens encapsulatedwithin droplets. Viability dyes have been used since the 1950's for cellviability purposes. However, these reagents are typically employed insamples that are significantly greater than 1 microliter in volumeand/or are used as an endpoint assay to indicate the presence of viablecells. Aspects of the invention use a viability dye in droplets that arebetween 1 pL and 100 nL, and more specifically 25-500 pL. In the methoddescribed here the optical signal generated by the viability dye isconcentrated by the small droplet volume and measured and recorded overan incubation time. In droplets containing viable cells, this results inan optical signature that is rapidly generated and has information aboutthe characteristics of the cell encapsulated within the droplets.Combined with an environment stressor, such as an antimicrobial orcytotoxic drug, an additional signature can be generated by monitoringthe optical signal of the droplets containing a cell over time. Theoptical signatures from the cell with and without the environmentalstressor can be used to determine the identity and/or characteristics ofthe cell. Furthermore, the differences between the optical signaturesobtained from a species of cells exposed to a drug compared to theoptical signatures for same species of target cells that are not exposedto the drug can be used to determine the phenotypic drug resistanceprofile for the target cells obtained from a test sample. Because thesesignatures are generated from individual cells encapsulated in droplets,they represent information about the individual characteristics of eachcell as opposed to an average characteristic of a population of cellsthat is generated from a bulk sample containing many cells.

The methods of the invention are compatible with any viability orreporter dye that can be used with live cells (does not require celllysis). In a preferred embodiment the viability dye is a resazurin-baseddye or derivative thereof. When blue, non-fluorescent resazurin isirreversibly reduced to pink and highly fluorescent resorufin (FIG. 6)it produces a fluorescent signal and a colorimetric shift (from blue topink). In a preferred embodiment, the fluorescence is used because itoffers better sensitivity over colorimetric signal changes. Thelimited-diffusion confinement within a sub-nanoliter volume of secretedfluorescent molecules quickly concentrates to detectable signal levelsand is then detected by the methods described below. Furthermore,resorufin is reversibly reduced to non-fluorescent hydroresorufin (FIG.6) if the redox environment dips below a particular redox threshold,usually around −100 mV. The combination of irreversible reduction fromresazurin to resorufin and the reversible reduction of resorufin tohydroresorufin and oxidation of hydroresorufin back to resorufindepending on the redox potential of the droplet are what create theunique fluorescence signature over time in droplets that are smallenough volume such that redox changes occur quickly in the presence of asingle cell or cell aggregate. Examples of commercially availableresazurin-based dyes are: AlamarBlue™ (various), PrestoBlue™ (ThermoFisher Scientific), Cell-titer Blue™ (Promega), or Resazurin sodium saltpowder. Dyes that are structurally related to resazurin and can be alsobe used in the method are: 10-acetyl-3, 7-dihydroxyphenoxazine (alsoknown as Amplex Red™) 7-ethoxyresorufin, and1,3-dichloro-7-hydroxy-9,9-dimethylacridine-2(9H)-one (DDAO dye). Inalternate embodiments dyes that rely on tetrazolium-reduction, such asformazan dyes, can be used as the cell viability indicator. Examplesinclude INT, MTT, XTT, MTS, TTC or tetrazolium chloride, NBT, and theWST series.

Cell (DCC) Aggregates.

A preferred application of the invention is towards the diagnosis ofmicrobial infections by identifying the microbes causing the infectionand whether or not they are resistant to antimicrobial drugs. Thus, inthis application, the DCCs can be single-celled microbes. Some bacteria,however, aggregate naturally into clusters or chains. In these cases,some droplets may comprise an aggregate of cells of the same microbialspecies (homogenous aggregate) rather than a single microbe. In thesecases, the shape of the curve may be affected by the number of cells inthe aggregate. However, the stored signature waveforms and call logicthat are used to classify the compartmentalized cells can account forsuch aggregates the same way they can account for single cells.Furthermore, if the embodiment includes antimicrobial susceptibilitytesting the mixture comprising the antimicrobial drug will exhibit thesame cell aggregation characteristics as the mixture that excludes theantimicrobial drug and the comparison will still be accurate. Therefore,while the method of the invention generally comprises isolation ofsingle-cells in each droplet, it necessarily accommodates the case of asingle cell species in a homogenous aggregate isolated in the dropletrather individual cells. In the case of cancer disease diagnosis, thetarget DCCs typically do not aggregate if they are circulating tumorcells. If the cancer cells are obtained from tissue, the tissue istypically disintegrated into individual cells prior to analysis.Therefore, each droplet will contain at most one cell; however, in someinstances a cancer aggregate may also be analyzed using the describedmethods.

Signal Detection.

Once the droplets have been generated, they must be presented foranalysis by an optical system, sensor, or sensor array. In a preferredembodiment, the droplets are presented in a two-dimensional array (FIGS.3, 4, and 5) so that good thermal control can be maintained and thedroplet signals can be measured simultaneously (at a single instance intime) for many droplets. In the droplets containing target cells, thereporter will produce a concentrated fluorescent signal that will riseabove the background droplets that do not contain cells (FIGS. 3, 4, and5). The concentrated signal of the droplet enables single cellidentification in comparable time standard PCR techniques which are thegold standard for fast identification. In certain aspects the signal isdetected by exciting a reduced reporter with a specific wavelength oflight and collecting the bandpass-filtered, Stokes-shifted light with acamera as shown in FIG. 4. The advantage to use imaging techniques isthat they can image a droplet array that remains stationary and cantherefore easily be monitored over time. Cytometry based methodstypically employ endpoint detection instead of real time detectionbecause of the difficulty in keeping track of the moving droplets overtime. Another advantage to imaging the array is that all the dropletsexperience the same reaction conditions at the time of analysis.Therefore, droplet signals can be compared at equivalent time pointswhich is important since signals vary over time. With a cytometryapproach, droplets pass by the detector at different times. Therefore,some droplets are incubated longer than others at the time of analysis.Finally, there may be different target cell species in the test sample.For each species, there may be an optimal droplet volume and dyeconcentration that maximizes signal at a particular time point. If anendpoint method is used, droplet volume and reporter concentrations donot need be controlled to the same degree because time can compensatefor sub optimality and different species can be characterizeduniversally within a single dye and droplet concentration.

Multiplexing.

The methods described herein include the specific identification ofmultiple cells from a single test sample. By compartmentalizing singlecells into their own isolated droplet, competition for resources betweencells is eliminated. Therefore, individual cells that would existcollectively as a minority in a bulk population, now have equal accessto nutrients when compared to the majority population of cells whichresults in a higher sensitivity for low abundance cells in a sample withmultiple cells types. The multiplexing limitations for this inventiondepend on the ability to differentiate viability signatures betweendifferent cell types. FIG. 8 is a schematic illustration showing twodifferent cell types in the droplets and a graph of the fluorescentsignals generated by two different bacterial species, E. coli and S.epidermidis, that were in the same test sample. Most methods formultiplexing require multiple dyes (fluorophores) which, in turn,require multiple sets of LEDs, excitation, and emission filters. Becausethe method described herein uses shape information rather than spectralinformation, the method can be used to multiplex many targets with asingle dye requiring only one LED, emission filter, and excitationfilter, thus simplifying the hardware needed to perform the analysis.

The preceding description and examples, as well as the figures areincluded to demonstrate particular aspects of the invention. It shouldbe appreciated by those of skill in the art that the techniquesdisclosed in the description, examples, or figures represent techniquesdiscovered by the inventors to function well in the practice of theinvention, and thus can be considered to constitute particular modes forits practice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

What is claimed:
 1. A method for detecting antimicrobial response ofmicrobes in a sample comprising: (a) contacting a sample comprisingmicrobes with a reporter forming a sample mixture; (b) dividing thesample mixture into at least two portions, wherein a first portion istreated with an antimicrobial substance forming a test portion and thesecond portion is a control portion; (c) partitioning the test portionand control portion into small volumes forming small volume partitionshaving one microbe or a natural aggregation of microbes per small volumepartition; (d) incubating the small volume partitions over time at aspecific temperature or temperatures; (e) monitoring opticalcharacteristics of the small volume partitions during the incubationtime, wherein the optical characteristics include the amount of opticalsignal produced by interaction of the reporter with the microbe orenvironment in the small volume partition; (f) constructing an opticalsignal waveform for each small volume partition resulting in a smallvolume partition waveform; and (g) comparing the optical signal waveformshapes of the small volume partitions derived from the control portionto the optical signal waveform shapes of the small volume partitionsderived from the test portion to determine the sensitivity of themicrobes to the antimicrobial substance.
 2. The method of claim 1,wherein the small volume partition waveform is compared to a library ofstored waveforms.
 3. The method of claim 1, wherein the small volumepartitions are droplets in an immiscible fluid.
 4. The method of claim3, wherein the immiscible fluid is a fluorocarbon comprising afluorosurfactant.
 5. The method of claim 1, further comprising arrangingthe small volume partitions in a two-dimensional array monolayer.
 6. Themethod of claim 5, wherein the two-dimensional array is a statictwo-dimensional array.
 7. The method of claim 3, wherein partitioningthe test portion and control portion is by Laplace pressure gradients.8. The method of claim 3, wherein partitioning the test portion andcontrol portion is by shear stress.
 9. The method of claim 1, whereinmonitoring optical characteristics of the small volume partitions isperformed using a camera.
 10. The method of claim 1, wherein thereporter is a fluorescent dye.
 11. The method of claim 1, wherein thereporter is a resazurin dye.
 12. The method of claim 1, where thereporter is a tetrazolium dye.
 13. The method of claim 1, wherein themonitoring optical characteristics of the small volume partitionsfurther comprises exciting the reporter using a light source.
 14. Themethod of claim 13, wherein the light source is a light emitting diode(LED).
 15. The method of claim 1, wherein the incubating of the smallvolume partitions is at a constant temperature.
 16. The method of claim15, wherein the constant temperature is 37° C.